Prolonged alpha/beta interferon (IFN-alpha/beta) or recombinant IFN-beta treatment of RS485, NIH 3T3 cells transformed by a long terminal repeat-activated Ha-ras proto-oncogene resulted in revertants that maintain a non-transformed phenotype long after IFN treatment had been discontinued. Cloned persistent revertants (PRs) produced large amounts of the ras-encoded p21 and were refractile to transformation by EJras DNA and by transforming retroviruses which carried the v-Ha-ras, v-Ki-ras, v-abl, or v-fes oncogene. Transient treatment either in vitro or in vivo with cytidine analogs that alter gene expression by inhibiting DNA methylation resulted in transformation of PR, but not of NIH 3T3 cells. We wish to study the mechanisms involved in this persistent reversion. We hope to demonstrate altered patterns of gene expression in the persistent revertants by identifying in cDNA libraries cell messages that are differentially expressed in PRs and RS485s. The potential of these cDNAs to affect the phenotype of RS485 or persistent revertant cells will be evaluated by transfecting these cells with constructs that will allow the over-expression of either message or anti-sense message. Genomic clones of differentially expressed gene will also be obtained and the flanking regulatory regions analyzed. We also wish to investigate the possible roles of co-transforming oncogenes, endogenous IFN production, and DNA methylation of regulatory sites in the phenomenon of persistent reversion, and to study further the resistance of PRs to re-transformation by various viral and cellular oncogenes.